PROJECT SUMMARY Sexually transmitted diseases (STIs) are the most frequently reported bacterial infections of humans in the United States, surpassing all other infections combined, including respiratory and enteric infections. Among STIs, Chlamydia trachomatis (CT) infections are the most frequently reported bacterial infections. CT co- infections with the gonococcus (GC), Neisseria gonorrhoeae, are relatively frequent. Together these pathogens are responsible for most cases of pelvic inflammatory disease (PID), which may lead to sequelae that profoundly impact female reproductive health: tubal factor infertility (TFI) and life-threatening ectopic pregnancy. With respect to GC infections, resistance to common antibiotics is a growing concern. While NAAT-based diagnosis of these 2 infections is highly specific and sensitive, it fails to address 2 major issues: 1) a positive diagnosis often necessitates that the patient returns for a second visit for treatment, and 2) currently available tests do not discriminate between infection and disease, a significant problem, particularly in the case of asymptomatic infections. Project 2 aims to identify serum biomarkers that can be diagnostic and/or predictive of CT infection and CT/GC co-infection. Serum is a convenient physiological sample that is easily and inexpensively obtained, is highly versatile with respect to laboratory testing, and relatively stable and easy to store. As such serum has been used extensively for a multiplicity of medical purposes with great success. Although serology has often been dismissed as a useful tool in the detection of CT and GC, we propose to develop two assay systems in Project 2 that will take serology to a new level where CT and GC are concerned. We will first use a highly specific ELISA system to detect antibody titers to a variety of outer membrane or secreted proteins of CT and GC that may be diagnostic or predictive of CT or GC infection and co-infection and subsequent pathology. Preliminary studies suggest that antibody responses to 3 CT outer membrane proteins are associated with PID. A second objective will be to identify microRNAs, small RNAs that are made by the host in response to a variety of stimuli, that may be exploited as specific biomarkers of CT or GC infection and co-infection and subsequent disease. While the overarching objective of this project is strongly translational, with the future development of highly sensitive and specific point-of-care and/or predictive tests for STIs and associated pathologies as the primary focus, it is also probable that our findings will uncover biomarkers that are revealing of underlying mechanisms of pathogenesis. Although these will not be investigated here, Project 2 findings will enhance and complement studies of STI pathogenesis undertaken in Projects 1 and 3 concerning the role of human genetic variance and host response, the role of the microbiota and the pathogenomics of CT and GC in the production of pathology.